Algae represent a potentially inexpensive, scalable, CO2-fixing, solar-powered source of diverse chemical products including biofuels, synthetic building blocks, nanomaterials, recombinant proteins, vaccines, antibodies, medicinal leads and food additives (Specht et al., (2010) Biotechnol. Letters 32: 1373-1383; Christenson &, Sims (2011) Biotechnol. Adv. 29: 686-702; Radakovits et al., (2010) Eukaryotic Cell 9: 486-501; Park et al., (2011) Bioresour. Technol. 102: 35-42; Mayfield et al., (2007) Curr. Opin. Biotechnol. 18: 126-133). They are also promising organisms for drug discovery and screening and have recognized value for bioremediation and as biosensors (Davis et al., (2003) 37: 4311-4330; Marshall WF (2009) J. Biomol. Screening 14: 133-141; Nagle & Zhou (2009) Phytochem. Rev. 8: 415-429). However, as encountered in the delivery of agents (e.g., siRNA and biologics) into mammalian cells, efforts to study or control the inner-workings of algal cells, as required for numerous research and commercial applications, are severely limited by problems encountered in the delivery of probes, genes and biomacromolecules across algal cell wall and membrane barriers. The delivery of chemical and biological agents into algal cells has been limited to physical and mechanical techniques (e.g. glass bead transfection, microinjection, electroporation, sonication, and biolistic methods) that are primarily used with cell wall-deficient mutants (Azencott et al., (2007) Ultrasound in Med. & Biol. 33: 1805-1817; Harris EH (2009) The Chlamydomonas Sourcebook, Second Edition. 1: 293-302; Kilian et al., (2011) 108: 21265-21269). While effective for many applications, these delivery methods are not scalable, show high variability within a given cell population, and can produce cellular damage and contamination (for instance, biolistic gold or tungsten particles). A molecular method to deliver, on variable scale, small molecules, probes, and biomacromolecules across the cell wall and membrane of wild-type algae, as required to probe and manipulate intracellular pathways in intact algae, would enable new opportunities in algal research and in the use of algae as photoautotrophic tools for synthetic biology. At the same time, such studies would serve to advance our understanding of biological barriers, a goal of central significance in the life sciences and agricultural and medical research.
It has been shown previously that the ability of guanidinium-rich molecular transporters (GR-MoTrs), including guanidinium-rich cell-penetrating peptides and non-peptidic agents, to enter mammalian cells is related to the number and spatial array of their guanidinium groups (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13003-13008). Subsequent studies have shown that GR-MoTrs enable or enhance the delivery of a variety of cargos, including small molecules, metals, imaging agents, iron particles, and proteins, into a variety of mammalian cell types (Wender et al., (2008) Adv. Drug Deliv. Rev. 60: 452-472; Wender et al., (2011) Drug Discovery Today: Technologies; Tung & Weissleder (2003) Adv. Drug Delivery Reviews 55: 281-294; Torchilin V P (2008) Adv. Drug Deliv. Rev. 60: 548-558). GR-MoTr-drug conjugates have also advanced to clinical trials for various indications including stroke, psoriasis, and ischemic damage (Johnson et al., (2011) Cell-Penetrating Peptides: Methods and Protocols, 535-551). Despite this progress on mammalian cells, little is known about the ability of GR-MoTrs to enter non-mammalian cells, especially those organisms of research and commercial significance which possess a cell wall. Only a few studies of GR-MoTrs with plant cells have been reported (Chang et al., (2005) Plant Cell Physiol. 46: 482-488; Chugh & Eudes (2007) J. Pept. Sci. 14: 477-481; Eggenberger et al., (2009) Chembiochem 10: 2504-2512; Chugh et al., (2009) Plant Cell Rep. 28: 801-810; Unnamalai et al., (2004) FEBS Letters 566: 307-310) and a single investigation of GR-MoTrs with algae (Liu et al., (2008) J. Membrane. Biol. 222: 1-15) However, in the latter study (Liu et al., (2008) J. Membrane. Biol. 222: 1-15), Chlorella vulgaris, a species of green algae with a cellulosic cell wall, was found to be impermeable to a GFP-nona-(L)-arginine fusion protein.